IN I LItti.\ I IONA I JOURNAI OF 1.1-TROSY^
Volume 48. Number 3
Printed in the U.S.A.
The Collagen of Permanently Damaged
Nerves in Human Leprosy'
Luiz C. U. Junqueira, Gregorio S. Montes, Estevam Almeida Neto,
Cecy Barros, and Antonio J. Tedesco-Marchese'
Studies of collagen have shown that at
least four different types of this protein exist in human tissues. Collagen type I is present in the (fermis, hone, tendon, and dentin:
collagen type II is found in hyaline and elastic cartilages, and collagen type Ill is present associated mainly with collagen type I
in arteries and in the uterus. Collagen type
IV has been described as a constituent of
basement membranes ("). Recent biochemical observations have shown the presence
of collagen types I and Ill in the human
femoral nerve (").
Results from this laboratory have shown
that the increase in hirefringency observed
in tissue sections, stained with Picrosirius
and studied with polarization microscopy,
is specific for collagen (1. Furthermore,
this method permits the distinction of collagen types I and 111 ("). Under these conditions, collagen type I appears as thick,
bright (strongly hirefringent), red or yellow
fibers. Collagen type Ill is characterized by
thin, pale (weakly birefringent), greenish
fibers. When applied to nerve sections, this
method localized collagen type Ill in the
endoneurium and collagen type I in the
epineurium 7 ).
Additional observations with electron
microscopy showed that collagen fibrils in
the endo- and perineurium have distinctly
smaller diameters than those of the collagen
fibrils in the epineurium. Nerves therefore
have two distinct collagen populations.
These populations can he characterized by
means of light and electron microscopy,
and they are localized in different structural
compartments of the nerve 8
(
(
).
' Received for publication on 11 December 1979:
accepted for publication on 14 May 1979.
I.. C. U. Junqueira, M.D.: G. S. Montes, D.V.Sc.,
1,aboratory of Cell Biology: E. Almeida Neto, M.D.,
C. Barros, M.D., Department of Tropical Medicine
and Dermatology: A. J. Tedesco-Marchese, M.D.
Department of Neuropsychiatry: University of S;io
Paulo, Faculdade de Nledicina, Avenida Dr. Arnaldo
455, 01246-Sto Paulo. Brasil.
Since leprous lesions are known to induce proliferation of collagen in nerves ('•
u
it was of interest to apply both the
l•
Picrosirius-polarization method and electron microscopic observation to peripheral
nerves from leprosy patients. Our observations strongly suggest that, in these lesions, collagen proliferation occurs in two
separate compartments with increases of
collagen type Ill in the peri- and endoneurium and of collagen type I in the epineurium.
'
),
MATERIALS AND METHODS
Clinical material. Segments of peripheral
nerves from ten chronically treated leprosy
patients, whose clinical diagnosis had been
confirmed by histopathological studies,
were used. These nerve segments were obtained in the course of clinically-indicated
surgical procedures involving affected portions of the nerves. We are dealing therefore with selected samples of leprous neuritis in all of which the pathologic changes
in the nerve lesions were characterized by
a proliferation of collagen (fibrosis) in the
three nerve compartments (endo-, epi-, and
perineurium).
Normal control nerves were obtained
from autopsies.
The Picrosirius-polarization method. Onehalf of the excised segment of each nerve
was fixed in Bouin's fluid for 24 hr, dehydrated, embedded in paraffin, and sectioned at 5 ium. These sections were stained
for one hr in 0. I% Sirius Supra Red F313A
as described C) and counterstained with
hematoxylin. The slides were studied with
an optical microscope fitted with polaroid
filters. To observe the thin pale fibers of
collagen type III, very strong illumination
was necessary. This was made possible by
overloading the voltage of the illumination
lamp.
Electron microscopy. The other half of
each biopsy was fixed in 2% glutaraldehyde
191
292^
International Journal of Leprosy^
in 0.1 M phosphate or cacodylate buffer for
two hr, followed by a second fixation in I%
osmic acid for one hr. 13lock staining was
performed in 0.5C; uranyl acetate overnight. The material was embedded in araldite, sectioned in a I.K13 ultratome, and
double stained in uranyl acetate and lead
citrate. Micrographs were obtained with a
Zeiss EM 9 S electron microscope.
The diameters of collagen fibrils were
measured in suitably enlarged micrographs
with a Bausch & Lomb measuring magnifier. The magnification of the microscope
was calibrated with a diffraction grating. At
least 200 measurements were made in four
electron micrographs in each case.
RESULTS
Light microscopy. The study of the nerve
biopsies with the Picrosirius-polarization
method showed a remarkable increase of
hirefringent material in the endo-, perk and
epineurium when compared to normal control nerves (Fig. 1). Since hirefringency is,
in this method, specific for collagen, the increase of hirefringent material denotes an
increase in the amount of collagen present
in the biopsies. Further observation of this
material disclosed the presence of several
thin parallel layers of collagen (hirefringent
material) concentrically disposed in transverse sections of the nerve, subdividing it
into an inner and an outer region (Figs. lb
and 3).
Polarization microscopy showed that two
different types of collagen could be clearly
distinguished in these nerves: one type was
formed by thin, pale (weakly hirefringent),
greenish fibers typical of collagen type III
('). In normal nerves, these collagen fibers
formed a delicate network surrounding the
nerve fibers. In the biopsies from leprosy
patients these collagen type III fibers appear in two sites. The first site corresponds
1980
to the endoneurium although its appearance
is different from that of normal nerves. In
this location the fibers appear as irregular
agglomerates of thin fibers that occupy
part, if not all, of the place originally occupied by the nerve fibers (Fig. 2). The second site corresponds to the perineurium,
which appears in most biopsies as the inner
portion of the parallel concentric layers described above. It is interesting to note that
the perineurium, which is not easily ohserved in normal nerves due to its thinness,
is clearly evident in most of the leprosy
biopsies studied as the result of collagen
proliferation in between the layers of perineural cells (Fig. 3).
The other type of collagen observed in
nerves is made up of thick, bright (strongly
hirefringent), yellow or red fibers. These
fibers correspond to collagen type I, which
is present in the epineurium of normal
nerves. The biopsies from leprosy patients
show a thickening of the epineurium due to
an increase in the amount of this type of
fibers (Fig. 1). In most of the biopsies from
leprosy patients, this nerve sheath can he
divided into a compact outer region and a
laminated inner region circumscribing the
perineurium.
Electron microscopy. The electron microscopic study confirmed the observations
made with the Picrosirius-polarization
method. The nerve biopsies from the leprosy patients showed compartments when
compared to normal control nerves (Fig. 4).
The study of our leprosy material showed
that as in normal nerves ( 5 ), two different
populations of collagen can be easily distinguished by the diameter of their fibrils (Fig.
5). A population of thinner fibrils with a
smaller average diameter was found in the
endo- and perineurium while a population
of thicker fibrils was characteristic of the
epineurium (Table I). The diameters of the
FIG. I. Photomicrographs of cross sections of normal (A) and leprous (13) nerves, stained with Picrosirius
and observed with polarization microscopy. All hirefringent structures are collagen. Observe the remarkable
increase of collagen induced by the leprous infection. ( x60)
FIG. 2. Photomicrographs of a leprous nerve stained by Picrosirius and observed with normal (A) and
polarization (13) microscopy. The epineuriurn (Er is formed by thick, bright collagen fibers showing a yellow or
red color when observed with polarization while the endoneurium (EN), is formed by pale, thin fibers that
present a greenish color. The perineural cell layers (I') are separated due to retraction during the histological
procedure. ( x 265)
48, 3^I/nu/tic/J.(1, et al.: Collte, eit in Leprosy Nerves^ 293
,
294^
intermit/0mi/ Journal of Leprosy
1980
48, 3^Itorqueira, et al.: Colldf, en in Leprosy Nerves^ 195
,
Flo. 5. Electromnicrographs of the thin collagen fibrils of the endoneurium (A) and thick collagen fibrils
from the epineurium Ili) at the same magnification, illustrating the presence of 2 different collagen populations
in leprous nerves. ( x60,000)
TABLE I. Average diameters (mean ± S.D.) of collto., en fibrils present in the club,and epineurium of nerves from human leprosy patients.
,
Case No.^Nerve studied^
Epineurium^Endoneurium
(nm)^ (rim)^ratio
1^radial cutaneous branch ^88.54 ± 11.26^47.78 ± 7.08^1.85
2^n uusculo-cutaneous^ 78.30 ± 9.83^58.62 ± 7.96^1.34
3^great auricular^
68.38 ± 6.83^38.25 ± 5.29^1.79
4^great auricular^
89.98 ± 10.89^57.23 ± 7.77^1.57
5^ulnar^
65.80 ± 5.84^32.67 ± 4.60^2.01
6^medial plantar^
66.20 It 5.89^36.48 ± 4.34^1.81
7^radial cutaneous branch^89.08 ± 6.60^46.73 :4: 4.95^1.91
8^palmar cutaneous branch^69.78 ± 6.84^36.31 -4- 6.67^1.92
9^ul nar^
72.52 -± 6.47^39.51 -4- 5.32^1.84
10^radial cutaneous branch^89.24 ± 9.47^53.28 -4- 8.83^1.67
FIG. 3. Leprous nerve stained by Picrosirius, and observed with polarization microscopy, showing the
thickened perineurium which presents several concentric layers of collagen in between the perineum! cells.
This is responsible for the marked increase in the width of the perineurium when compared to a normal nerve.
The epineurium (right) shows characteristic, thick, bright fibers which contrast with the thin, pale fibers of the
endoneurium (left). ) x435)
FRi. 4. Electronmicrograph of a leprous nerve showing a marked increase in the endoneural collagen.
( x 7800)
International Journal of Leprosy ^
296^
1980
TABLE 2. Comparison between the averdfx diameters (mean S.I).) of collagen libril.s
from the endo- and epineurium of normal and leprous nerves.
Normal nerves"
Leprous nerves''
Number^Epineurium
of cases^(nm)
Endoneurium
(nm)
Epi-/endoratio
75.43^(- 7.83
77.78 ± 7.99
50.01^± 4.24
44.68 ± 6.28
1.51
1.74
5
10
.
Average of two ulnar, two femoral, and one musculo-cutaneous nerve. Data from Junqueira, el al. (")
'' Average of Table I.
thin and thick fibrils present in the biopsies
from leprosy patients were similar to the
values obtained from normal nerves (Table
types 1 and III in the endo-, perk and epineurium remains the same as in normal
nerves.
2).
DISCUSSION
Our results confirm those in the literature
0.2.4.12),
showing that leprosy lesions in
nerves promote an increase in their collagen content. They show that despite the
changes induced by this disease, the distribution of collagen types I and Ill in the
nerve compartments remains the same as
observed in normal controls.
Several lines of evidence suggest that
Schwann cells are the site of collagen production in the endoneurium ( 7 ) and that the
collagen produced is type 111 (s). This
agrees with Job and Verghese ( .1 ), who suggested that Schwann cells play an important role in the production of collagen in
leprous nerves. The fact that Schwann cells
are the preferential site of invasion by Mycobacterium l eprae (2.11, promoting leprous neuritis characterized by the proliferation of collagen, shown in the present
paper to be type III, supports the abovementioned contention.
The results of our study on the collagen
of leprous nerves show that despite the remarkable increase in the amount of this
protein, its other characteristics such as fibril diameters, localization, and types of
collagen present show no difference when
compared to normal nerves.
RESUMEN
Se estuditt el col:igen() de los nervios obtenidos de
diez pacientes leprosos titilizando el inetodo de Picrosiritis-polarizackin, y la microscopic electrimica. I'udo
comprobarse que la lepra produce tin notable Meremento del contenido de colageno en el nervio. A pesar
de los canibios provocados por esta enfermedad, la
localitticiin del colageno de los tipos I y III en el
endo-, perk y epineuro, se mantiene igual a la de los
nervios normales.
RESUME
Le collagens a etc etudie dans des biopsies nerveuses de dix cas de lepre par la methode du Picrosirius-polarisation et la microscopic clectronique. On a
observe que la lepre produit tine augmentation notable
de la quantite de collagene dans le nerf. Malgrc les
changements produits par cette maladie, la localisation
du collagens des types I et III dans l'endo-, perk et
epinevre reste semblable a cells des nerfs TIONTIMIX.
Acknonr ledgements. This study was undertaken with
a grant from the Fundacao de Amparo a Pesquisa do
Estado de Sao Paulo. We wish to thank I. A. Shishido,
K. M. Shigihara, and R. M. Kriszttin for technical
assistance.
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